The long-term objective of our investigations is the elucidation of the relationship between the chemical, conformation, receptor binding and biological activity of insulin and the relationship between the biological activity of insulin and the autophosphorylation and protein kinase activity of its receptor. Our studies thus far led to the synthesis of a new generation of insulins that have the potential to become better therapeutic agents than natural insulin in the clinical treatment of diabetes. We have synthesized superactive insulins (several-fold more active than the natural human hormone) which also display tissue specificity, i.e. they are more active in adipose tissue (peripheral system than in the liver. We have also synthesized insulins which appear to be hepatospecific, i.e. they are more active in the liver than in adipose tissue. Within this framework our specific aims include: (1) the synthesis of superactive insulin analogues; (2) the synthesis of superactive hepatospecific insulin analogues; (3) investigation of the structural features required for hepatospecificity of insulin analogues; (4) investigation of the affects of the structural modifications of the insulin molecules that lead to superactivity on the insulin structure; (5) investigation of the solution structure(s) of insuli and insulin analogues having special photophysical properties (by means of selective substitution of amino acid residues with fluorescent probes) by time-resolved fluorametry, relative changes in lifetimes, anisotrophy decays and energy transfers as a function of temperature, pH, fluorescence quenchers and chemical denaturants will be used to generate a structural perturbation map of the insulin molecule; (6) investigation of the link between biological activity of insulin and autophosphorylation and protein kinase activity of its receptor upon binding of insulin. Toward this end we will investigate the relationship between concentration dependence of analogue- stimlated autophosphorylation and kinase activity of various insulin analogues; in addition we will determine by means of phosphopeptide maps whether any difference exists in the sites of insulin- vs. analogue-stimulated receptor autophosphorylation.